Primarily due to differences in the cytokine environments to which macrophages are exposed, these can develop into different subsets exhibiting different functional and molecular properties (Goerdt and Orfanos, 1999; Gordon, 2000). The best-studied macrophage subsets are classically activated macrophages (caMF), differentiating in the presence of stimuli such as IFN-γ and LPS and being important components of host defense in the fight against various pathogens. On the other hand, type II cytokines such as IL-4 and IL-13, antagonize caMF and induce the development of alternatively activated macrophages (aaMF). The latter are considered to secure the balance between pro- and anti-inflammatory reactions during type I cytokine-driven inflammatory responses and to be involved in angiogenesis and wound healing (Goerdt and Orfanos, 1999). However, the association of aaMF with type II cytokine-controlled inflammatory diseases (Loke et al., 2000) suggests that, under these circumstances, aaMF may support the development of pathology. To gain better insights into the exact functional properties of aaMF in vivo, there is an urgent requirement for better and additional markers for (in situ) analysis of aaMF, as well as for FACS analysis of isolated cells. Discrimination between murine caMF and aaMF has so far been based mainly on differential arginine metabolism. Hence, in caMF, L-arginine is converted in NO and L-citrulline via inducible nitric oxide synthase (iNOS), whereas aaMF are characterized by an alternative metabolic pathway of arginine, catalyzed by arginase 1, converting L-arginine to L-ornithine and urea.
Recently, several new markers have been described. Gratchev et al. (2001) applied an in vitro approach, using a combination of subtractive hybridization and differential hybridization with IL-4- or IF-γ-stimulated macrophages, and found that fibronectin and the extracellular matrix protein βIG-H3 are differentially expressed. However, the in vitro stimulation may lead to artifacts. Therefore, Raes et al. (2002) used suppression subtraction hybridization in an in vivo experimental model of murine trypanosomosis to identify genes that are differentially expressed in aaMF versus caMF, and identified the secreted factors FIZZ1 and Ym as possible markers. Loke et al. (2002) constructed a subtractive library from purified peritoneal macrophages from Brugia malayi-implanted WT mice against peritoneal macrophages of IL-4−/− mice infected with B. malayi, and identified several possible marker genes, including the previously in vivo-identified FIZZ1 and Ym, but not the in vitro-identified markers.
Notwithstanding these newly identified marker genes, there is still a need for an easily identifiable marker. Surprisingly, we found that galactose C-type lectins 1 and 2 are also differentially expressed between aaMF and caMF. As these markers are situated on the cell membrane, they allow non-destructive recognition of the aaMF. Moreover, contrary to secreted markers, they allow, for the first time, a reliable sorting and purification of the aaMF fraction.